An abietane diterpenoid is a potent activator of systemic acquired resistance

Abietane diterpenoids are major constituents of conifer resins that have important industrial and medicinal applications. However, their function in plants is poorly understood. Here we show that dehydroabietinal (DA), an abietane diterpenoid, is an activator of systemic acquired resistance (SAR), which is an inducible defense mechanism that is activated in the distal, non-colonized, organs of a plant that has experienced a local foliar infection. DA was purified as a SAR-activating factor from vascular sap of Arabidopsis thaliana leaves treated with a SAR-inducing microbe. Locally applied DA is translocated through the plant and systemically induces the accumulation of salicylic acid (SA), an important activator of defense, thus leading to enhanced resistance against subsequent infections. The NPR1 (NON-EXPRESSOR OF PR GENES1), FMO1 (FLAVIN-DEPENDENT MONOOXYGENASE1) and DIR1 (DEFECTIVE IN INDUCED RESISTANCE1) genes, which are critical for biologically induced SAR, are also required for the DA-induced SAR, which is further enhanced by azelaic acid, a defense priming molecule. In response to the biological induction of SAR, DA in vascular sap is redistributed into a SAR-inducing 'signaling DA' pool that is associated with a trypsin-sensitive high molecular weight fraction, a finding that suggests that DA-orchestrated SAR involves a vascular sap protein(s).     

Identification of a diverse synthetic abietane diterpenoid library and insight into the structure-activity relationships for antibacterial activity

A diverse natural product-like (NPL) synthetic abietane diterpenoid library containing 86 compounds were obtained and the SARs were studied based on their antibacterial potential. Further in vitro cytotoxic and in silico drug-like properties evaluation showed that the potent antibacterial compound 84 had good drug-like properties and displayed low cytotoxicity toward noncancerous mammalian cells, indicating the study of AA and DHAA might be a good starting point for the search of novel antimicrobial molecules. Future work should be focused on the optimization of their potency and selectivity.     

Identification of a diverse synthetic abietane diterpenoid library for anticancer activity

A diverse natural-product-like synthetic abietane diterpenoid library contains about 56 compounds were obtained, and evaluated for their potential in vitro cytotoxic or antitumor activity against A549, PC-3 and SKOV-3 tumor cell lines by SRB assay. Treatment of A549 cells with the most potent compound ketone 19 showed induction of apoptosis, as revealed by JC-1 mitochondrial membrane potential staining, TUNNEL assay, western blotting analysis and flow cytometry assay.     

Rearranged abietane diterpenoid hydroquinones from aerial parts of Ajuga decumbens Thunb

Four new rearranged abietane diterpenoid hydroquinones, ajudecumins A–D (1–4), together with two known rearranged abietane diterpenoids, three neo-clerodane diterpenoids, four megastigmane derivatives, two flavonoids as well as a bisabolene sesquiterpenoid were isolated from the aerial parts of Ajuga decumbens. Their structures were established on the basis of extensive spectroscopic analysis and the stereochemistry of 1 was confirmed by single-crystal X-ray diffraction analysis. Among the diterpenoids, compounds 1 and 3 exhibited moderate inhibitory activity on the proliferation of human breast cancer MCF-7 cells.     

Fate of a bioactive fluorescent wortmannin derivative in cells

Here, we report on NBD-Wm, a fluorescent wortmannin (Wm) probe that maintains the bioactivity of Wm as an inhibitor of PI3 kinase and as an antiproliferative agent. The attachment of the NBD fluorochrome permits NBD-Wm in cells to be monitored by NBD fluorescence-based methods such as FACS or fluorescence microscopy or with an anti-NBD antibody. The fluorescence of NBD-Wm treated cells reached a peak at 1.5 h and then decreased because of the extrusion of a fluorescent compound into the culture media. Cells accumulated NBD-Wm to levels about 30-fold higher than those in the media. NBD-Wm modified five major proteins, with the modification of the catalytic subunit of PI3 kinase being a minor band. The bioactivity of NBD-Wm, coupled with a variety of techniques available for determining its disposition, suggest that NBD-Wm can be a useful tool in understanding the mechanism of action of viridins.     

An efficient conversion of 5-nitroisatin into 5-nitroindole derivative

Our process research on OPC-35564 revealed that a mixed borohydride reducing agent (ZrCl4/NaBH4) in DME (Itsuno system) afforded a rapid and direct conversion of N-alkyl-nitroisatin into nitroindole nucleus. Comparison with other reducing agents indicated the superiority of the present system and the key function of ZrCl4. For the manipulation of base-labile isatin, a useful procedure for its N-alkylation using Cu2CO3 is also presented.     

An easy way to include weak alignment constraints into NMR structure calculations

We have recently shown that an energy penalty for the incorporation of residual tensorial constraints into molecular structure calculations can be formulated without the explicit knowledge of the Saupe orientation tensor (Moltke and Grzesiek, J. Biomol. NMR, 1999, 15, 77–82). Here we report the implementation of such an algorithm into the program X-PLOR. The new algorithm is easy to use and has good convergence properties. The algorithm is used for the structure refinement of the HIV-1 Nef protein using 252 dipolar coupling restraints. The approach is compared to the conventional penalty function with explicit knowledge of the orientation tensor's amplitude and rhombicity. No significant differences are found with respect to speed, Ramachandran core quality or coordinate precision.     

A fluorescence study of the interaction and location of (+)-totarol, a diterpenoid bioactive molecule, in model membranes

(+)-Totarol, a diterpene extracted from Podocarpus totara, has been reported as a potent antioxidant and antibacterial agent. Although the molecular mechanism of action of this hydrophobic molecule remains unknown, recent work made in our laboratory strongly suggests that it could be lipid-mediated. Since (+)-totarol contains a phenolic ring, we have studied the intrinsic fluorescent properties of this molecule, i.e., quantum yield, lifetime, steady-state anisotropy and emission spectra, both in aqueous and in phospholipid phases, in order to obtain information on the interaction and location of (+)-totarol in biomembrane model systems. The phospholipid/water partition coefficient of (+)-totarol was found to be very high (K(p)=1.8x10(4)), suggesting that it incorporates very efficiently into membranes. In order to estimate the transverse location (degree of penetration) of the molecule in the fluid phase of DMPC model membranes, the spin labelled fatty acids 5-NS and 16-NS were used in differential quenching experiments. The results obtained show that (+)-totarol is located in the inner region of the membrane, far away from the phospholipid/water interface. Since (+)-totarol protects against oxidative stress, its interaction with an unsaturated fatty acid, trans-parinaric acid, was studied using fluorescence resonance energy transfer. No significant interactions were observed, molecules of trans-parinaric acid distributing themselves randomly amongst those of (+)-totarol in the phospholipid membrane.     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).     

A MAS-NMR study of the location of (+)-totarol,a diterpenoid bioactive molecule,in phospholipid model membranes

(+)-Totarol, a diterpenoid isolated from Podocarpus spp., is a potent antioxidant and antibacterial agent. Although the mechanism of action of this hydrophobic molecule is poorly understood, recent work from our laboratories suggests that it could be due to membranotropic interactions. The location of (+)-totarol in membranes and its interaction with membrane components is therefore of considerable interest. High resolution magic angle spinning (MAS) natural abundance 13C nuclear magnetic resonance studies were undertaken to assess the location of (+)-totarol in model membranes composed of egg yolk phosphatidylcholine (EYL). 13C spin-lattice relaxation times (T(1)) of both the phospholipid and (+)-totarol molecules in the presence of Gd(3+) were measured to obtain information on molecular distances. Our results indicate that (+)-totarol is situated in the upper region of the membrane, with its hydroxyl group located in the vicinity of the C-3/4 carbon atoms of the phospholipid acyl chain, and nearly perpendicular with respect to the phospholipid acyl chain axis. Such a location of (+)-totarol in the membrane would be expected to compromise the functional integrity of the membrane and account, at least in part, for its antibacterial effects.     

Isolation of a new bioactive cinnamic acid derivative from the whole plant of Viola betonicifolia

A new cinnamic acid derivative was isolated from the whole plant of Viola betonicifolia as off white needle. On the basis of various modern spectroscopic techniques including HREI–MS and 1D and 2D NMR, its structure was elucidated as 2,4-dihydroxy, 5-methoxy-cinnamic acid. It showed marked inhibition against DPPH (diphenyl-2-picryl hydrazyl) free radicals with IC₅₀ = 124?±?5.76 µM. The antioxidant property of the compound was compared with α-tocopherole and vitamin C having IC₅₀ values 96?±?0.46 and 90?±?0.56 µM, respectively. In case of antiglycation assay, the compound exhibited moderate activity (IC₅₀ = 355?±?7.56 µM) similar to standard compound, rutin (IC₅₀ = 294?±?0.56 µM). However, it was non-toxic to PC-3 cell line. It is concluded that 2,4-dihydroxy, 5-methoxy-cinnamic acid has antiglycation potential which was further augmented by its antioxidant activity and thus offered an ideal natural therapeutic option for the effective management of diabetes.     

An unexpected rearrangement giving a new thiosubstituted carbohydrate

A new thiosubstituted ‘d-arabino’-type derivative was obtained from an open carbohydrate via a cascade of four consecutive transformations in a single reaction process. Molecular orbital computations were also performed to explain the stereochemical outcome of the reaction.     

An easy assay for histone acetyltransferase activity using a PhosphorImager

A simple radiometric assay for histone acetyltransferase (HAT) activity employing a PhosphorImager is described. In the proposed procedure, following incubation of [1-¹⁴C]acetyl coenzyme A (CoA), histones, and HAT enzyme, radiolabeled histones are fixed on GF/F glass microfiber filter while the excess of acetyl CoA is washed out. Afterward, the filter is exposed to a phosphor-screen and the resulting spot signals are quantified with a PhosphorImager. Given the small volumes required, the new assay reduces reagent consumption and contaminated waste. Moreover, the assay can be performed with a large number of samples simultaneously, is applicable on different protein substrates, and is adaptable to the analysis of other protein modifications.     

Study on the interaction between human serum albumin and a novel bioactive acridine derivative using optical spectroscopy

The interaction of a novel bioactive agent N‐{[N‐(2‐dimethylamino) ethyl] acridine‐4‐carboxamide}‐α‐alanine [N‐(ACR‐4‐CA)‐α‐ALA] with human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption and circular dichroism spectrophotometric techniques under simulative physiological conditions. The fluorescence quenching of HSA by addition of N‐(ACR‐4‐CA)‐α‐ALA is due to static quenching and hydrogen bonding. Moreover, hydrophobic interactions play a role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA as well. The number of binding sites, n, and the binding constant values, K_A, were noted to be 0.88 and 3.4 × 10⁴ L mol^?1 for N‐(ACR‐4‐CA)‐α‐ALA at 293 K. The binding distances and the energy transfer efficiency between N‐(ACR‐4‐CA)‐α‐ALA and protein were determined. The negative value of enthalpy change and positive value of entropy change in the present study indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA. Copyright © 2010 John Wiley & Sons, Ltd.     

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor ent-kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the ent-kaurene overproducers in a closed environment, the airborne ent-kaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in ent-kaurene oxidase). These results suggest that ent-kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that ent-kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.     

Lipase-catalyzed production of a bioactive fatty amide derivative of 7,10-dihydroxy-8(E)-octadecenoic acid

Enzymatic syntheses of fatty amides are of considerable interest due to their wide ranging industrial applications in detergents, shampoo, cosmetics and surfactant formulations. Amidation reaction of Candida antarctica lipase B (CALB) was investigated for direct amidation of carboxylic acid in organic solvent. CALB-mediated production of a novel secondary amide was carried out by reacting the hydroxy oleic acid derivative, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), with N-methylethanol amine in organic solvent medium. A single, new product peak corresponding to the secondary amide of DOD (D2AM) was detected by high-performance liquid chromatography and thin-layer chromatography. The production of D2AM was achieved in high yields (95%) after 72 h at 50 degrees C in a CALB-catalyzed reaction that contained 100 IU enzyme activity, 50 mM DOD, and 100 mM N-methylethanol amine in isoamyl alcohol. The new fatty amide D2AM displayed potent antimicrobial activity towards Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Proteus vulgaris and Klebsiella pneumonae). D2AM also exhibited antioxidative activity by its alpha,alpha-diphenyl-beta-picryl-hydrazyl (DPPH) radicals scavenging effects.     

Incorporation of alpha-amylase enzyme and a bioactive filler into hydrophilic, partially degradable, and bioactive cements (HDBCs) as a new approach to tailor simultaneously their degradation and bioactive behavior

Hydrophilic, partially degradable, and bioactive cements (HDBCs) are starch-containing cements intended to degrade partially in the human body and, in so doing, allow for bone ingrowth inside the pores formed during degradation. Therefore, the study of degradation and bioactivity behavior was performed to assess the suitability of the current HDBCs formulations to achieve those aims. The degradation profile of HDBCs was studied under different conditions, including incubation in phosphate-buffered saline (PBS) and PBS supplemented with alpha-amylase at different concentrations. Thermostable alpha-amylase was also added to some formulations to allow control of the degradation rate and its extent. In a second stage the simultaneous phenomena of enzymatic degradation and bioactivity (both in vitro) was studied. We observed that the degradation of starch present in HDBCs can be easily controlled by the amount of alpha-amylase added to the cement and high values of degradation may be achieved if high enough quantities of enzyme are incorporated. However, the maximum degradation extent is much more dependent on the total amount of starch present in the formulation than on the amount of enzyme added to it: for full pore connectivity, the amount of starch should be higher than the percolation threshold for a 3D specimen. Nonetheless, calcium phosphate was able to nucleate and spread in inner pores of the cement, formed due to degradation, if they were interconnected. For a more thorough covering of the pores with calcium phosphates the amount of starch present in HDBCs should be increased to be higher than the percolation threshold.     

Antimalarial activity of abietane ferruginol analogues possessing a phthalimide group

The abietane-type diterpenoid (+)-ferruginol, a bioactive compound isolated from New Zealand’s Miro tree (Podocarpus ferruginea), displays relevant pharmacological properties, including antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and anticancer. Herein, we demonstrate that ferruginol (1) and some phthalimide containing analogues 2–12 have potential antimalarial activity. The compounds were evaluated against malaria strains 3D7 and K1, and cytotoxicity was measured against a mammalian cell line panel. A promising lead, compound 3, showed potent activity with an EC₅₀ = 86 nM (3D7 strain), 201 nM (K1 strain) and low cytotoxicity in mammalian cells (SI > 290). Some structure–activity relationships have been identified for the antimalarial activity in these abietane analogues.